Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010-05-19 to 2010-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliabe without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
modified OECD 429, method according to Ehlings et al. 2005
Principles of method if other than guideline:
The test was performed in accordance with the method according to Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ammonium thiosulfate
- Molecular formula: (NH4)2S2O3
- Molecular weight: 148.20 g/mol
- Physical state: Solid, white powder
- Storage condition of test material: At room temperature in a tightly closed container.
- Melting point: n.a.
No further information on the test substance was stated.

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Approx. 9 weeks
- Weight at study initiation: 27 - 31 g
- Housing: The animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C +/- 3°C (maximum range)
- Relative humidity: 55% +/- 15% (maximum range)
- Air changes: 12 - 18 times per hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Study design: in vivo (LLNA)

Vehicle:
other: aqua ad iniectabilia
Concentration:
10 % w/w, 25 % w/w, and 50 % w/w of ammonium thiosulfate
No. of animals per dose:
6 female mice
Details on study design:
RANGE FINDING TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10%, 25% and 50% of ammonium thiosulfate in aqua ad iniectabilia were examined.
Results:
In a preliminary experiment, concentrations of 10%, 25% and 50%, employing 1 animal per concentration, were examined. No pronounced irritating properties were observed in this preliminary experiment at concentrations of 10%, 25% or 50%, no differences in ear weight and ear thickness were noted.

MAIN STUDY
The test item had to be dissolved in aqua ad iniectabilia (Batch no. 911728; Delta Select, 63303 Dreieich, Germany). Due to the chemical properties of ammonium thiosulfate no suitable homogeneous dosage form could be obtained with less polar, aprotic solvents.

The test item solution was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.

The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS:
The following observations were made during the course of the study:
- Clinical signs: Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous mem¬branes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
- Body weight: The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS:
The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the NALIMOV test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.
No further information on the study design was stated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "Details on study design".

Results and discussion

Positive control results:
The positive control group caused the expected increases in lymph node cell count (S.I.: 1.986) and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study is regarded as valid. The stimulation index for lymph node weight was 1.844 and for ear weight 1.198.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method). 10 % w/w: SI: 0.885 (cell count); SI: 0.951 (lymph node weight); SI: 1.008 (ear weight) 25 % w/w: SI: 0.546 (cell count); SI: 0.732(lymph node weight); SI: 0.977 (ear weight) 50 % w/w: SI: 0.940 (cell count); SI: 0.927 (lymph node weight); SI: 1.038 (ear weight)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Any other information on results incl. tables

Treatment with ammonium thiosulfateat concentrations of 10%, 25% or 50% did not reveal statistical significantly increased values forlymph nodecell count, all stimulation indices for the lymph node cell count were beneath the threshold value of 1.4. In addition, the lymph node weight was not increased. Hence, the test item is classified as not sensitising.

The threshold level for the ear weight of 1.1 was not exceeded, i.e. no irritating properties were noted.

(Please refer to "Attached background material" for the raw data on stimulation indices.)

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the present test conditions, ammonium thiosulfate at concentrations of 10%, 25% and 50% (w/w) in aqua ad iniectabilia did not reveal any sensitising properties in the local lymph node assay. Therefore, the test item must not be classified and labelled according to regulation (EC) No.: 1272/2008.
Also, according to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as skin sensitiser.